Process of using prodigiosin as an immunosuppressive

ABSTRACT

There are disclosed a novel microorganism  Serratia marcescens  strain and a prodigiosin isolated from the microorganism. The prodigiosin is useful as an immunosuppressive in various fields, including the treatment of the diseases requiring immunosuppression and the basic research for the diseases, the transplantation of the organs or tissues, and the immune cells.

TECHNICAL FIELD

The present invention relates to a novel Serratia marcescens strain, aprodigiosin, and the use of the prodigiosin in immunosuppression fields.More particularly, the present invention relates to a novel Serratiamarcescens strain which can produce the prodigiosin, and the use of theprodigiosin as an immunosuppressive.

BACKGROUND ART

Over the recent few years, active study and research have been andcontinued to be directed to the development of immunosuppressives, whichare useful for the study on immunocytes and immune responses and for thetreatment of the diseases requiring immunosuppression. For instance,immunosuppressives are utilized in researching almost all of immuneresponses, including cytokine production, T-cell activation, antibodyproduction, cell death, DNA synthesis, immunocyte differentiation,intracellular signal transduction, etc. The immunosuppressives are alsoused to treat the diseases attributable to exaggerated immune responses,such as hypersensitive immune response and allergies. In addition, theyare needed to suppress excess immune responses upon transplantation oforgans, such as the kidney, the liver, the pancreas, marrow, the heart,skin, the lung, etc.

Prevailing immunosuppressives include, for example, cyclosporin A,cyclophosphamide, rapamycin, FK-506, etc. Many immunosuppressives whichshow similar or different suppressing behaviors are now under research.

The microorganisms belonging to genus Streptomyces or Serratia producered substances of pyrrolylpyromethene structures, examples of whichinclude prodigiosin, metacycloprodigiocin, prodigiosene,methoxyprodigiosin, and prodigiosin 25-C. They are now known to be ofantibacterial and antimalarial activity and, particularly, prodigiosin25-C shows an immunosuppressing effect.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a novel strainSerratia marcescens which produce a prodigiosin.

It is another object of the present invention to provide a prodigiosinas an immunosuppressive.

BEST MODES FOR CARRYING OUT THE INVENTION

The detailed description of the present invention will follow isolationof a desired microorganism strain; mycological characterization of thestrain; extraction of prodigiosin with organic solvent; purification ofprodigiosin through silica gel column and thin layer chromatography;structure analysis through nuclear magnetic resonance; utility of theprodigiosin as an immunosuppressive.

Germ-free test animals, mice BDF1 and B6C3F1, obtained from GeneticResources Center, Korean Research Institute of Bioscience andBiotechnology in the Korean Institute of Science and Technology, wereused for the assay of the immunosuppressive activity of prodigiosin. Thedata from the ex vivo experiments concerning the immunosuppressiveeffect of prodigiosin show that as much as 300 nM of prodigiosin has acytotoxic effect, but no effects at less than 100 nM. At suchconcentrations as show no cytotoxic effects, prodigiosin cannot suppressthe immune response of B lymphocytes. Prodigiosin had no influence onthe antibody production and proliferation of B lymphocytes, but has apotential suppressive effect on the proliferation and activity of Tlymphocytes. This selective immunosuppression for T lymphocytes is notascribed to the selective cytotoxicity for T lymphocytes. The sameimmunosuppression results as in the ex vivo experiments were obtained inin vivo experiments. When T lymphocyte activity was measured by use of agraft versus host reaction and a T cell-dependent antibody producingreaction, the prodigiosin suppressed the immune response, but exerted notoxicity on animals. Therefore, the immunosuppressive activity of theprodigiosin is thought to be attributed to the selective suppression forT lymphocyte activity.

Prodigiosin 25-C, an immunosuppressive analogous to, but different fromprodigiosin in structure and molecular weight, is known to suppress theproliferation of T lymphocytes, but not the proliferation of Blymphocytes. Of T lymphocytes, CD8 T lymphocytes are suppressed, but CD4T lymphocytes are not. In contrast, the prodigiosin of the presentinvention has an immunosuppressive activity on CD8 T lymphocytes and CD4T lymphocytes, both. This immunosuppressive activity is similar to thoseof other preexisting immunosuppressives. Like commercially availableimmunosuppressives, such as Cyclosporin A, Cyclophosphamide, FK-506 andRapamycin, the prodigiosin of the invention selectively suppress theimmune response of T lymphocytes.

The reaction systems used in the present invention are illustrative ofthe application of prodigiosin for a basic research of immunology, butnot limitative of the use of prodigiosin. The immunosuppressives incurrent use are needed in various fields. First of all, the treatment ofthe diseases requiring immunosuppression and the basic research thereforrequire them. Immunosuppressive drugs are useful to remove the immuneresponse which follows the transplantation of organs or tissues. Anotherapplication field of immunosuppressives is a basic research related toimmune cells. In this field are included studies on cytokines,activation and differentiation of immune cells, and intracellular signaltransduction. Cyclosporin A, Cyclophosphamide, FK-506 and Ripamycin areavailable for this field. Because the prodigiosin of the presentinvention has an activity similar to that of the aboveimmunosuppressives, it can be used as a curing agent and a standard insuch various fields.

The prodigiosin of the present invention was found to have the followingchemical formula with a molecular weight of 323 as measured by NMR.

A better understanding of the present invention may be obtained in lightof the following examples which are set forth to illustrate, but are notto be construed to limit the present invention.

EXAMPLE I

Culturing of a Serratia marcescens Strain and Isolation of Prodigiosin

Soil samples were taken from a silt area in Mokpo, Korea. A bacterialgroup belonging to Serratia spp. was isolated from the samples and namedSerratia marcescens B-1231. It was deposited in Korean Collection forType Cultures, Korean Research Institute of Bioscience and Biotechnologyon Sep. 19, 1997 and received a Deposition No. KCTC-0386BP. In order toobtain an immunosuppressive, the Serratia marcescens B-1231 was culturedat 28° C. for 62 hours in a 1 L Erlenmeyer flask containing a basicmedium which consisted of soluble starch 1%, phamamedia 0.5%, glucose0.2%, ammonium sulfate 0.1%, potassium phosphate 0.1%, MgSO₄.7H₂O 0.05%,calcium chloride 0.1% and NaCl 0.3%, at pH 7.0. An equal amount of ethylacetate was added to the culture and they were sufficiently mixed for 30min to give an organic layer. As the organic layer was concentratedunder a reduced pressure, a red substance was obtained. This wasseparated by silica-gel column chromatography using as a mobile phase amixture of chloroform and methanol. Following this, silica gel thinlayer chromatography was carried out to purify the object material.

EXAMPLE II

In vitro Experiment for Cytotoxicity Effect of Prodiaiosin onLymphocytes

Immune cells were separated from the spleens of the germ-free animalsand cultured in vitro. The cultures were treated with the prodigiosin atvarious amounts from 3 nM to 30,000 nM and the viability of the cellswere measured from the first day to the third day after the treatment.Based on the initial viability of the immune cells, the viabilities ofthe test groups were calculated. The results are given as shown in Table1, below. As apparent from the data, the viability of the treated immunecells is significantly decreased at a concentration not less than 300 nMwhen being compared with that of an untreated control. So, subsequentexperiments for immunoactivity were carried out at not more than 100 nMin order to exclude the cytotoxicity and to measure only theimmunosuppressive effect of the prodigiosin.

TABLE 1 Effect of Prodigiosin on the Viability of Immune Cells Conc. ofProdigiosin Viability (%) Groups (nM) 1st day 2nd day 3rd day Non- 93 7977 treated Treated 3 96 86 79 10 89 82 79 30 89 70 81 100 82 70 70 30068 14 18 1,000 74 14 14 3,000 61 9 8 10,000 32 4 4 30,000 4 4 4

EXAMPLE III

In vitro Experiment for the Effect of Prodiaiosin on Immune CellProliferation

Three standard substances which induce lymphocytes to proliferate wereemployed to measure the effect of the prodigiosin on proliferation oflymphocytes. 5 μg/ml of lipopolysaccharide were used to induce Blymphocyte to proliferate, 5 μg/ml of Concanavalin A for T lymphocyteand 5 μg/ml of Pokeweed mitogen for B and T lymphocytes, both.Prodigiosin was added, together with the proliferation-inducingsubstance. Three days after the addition, the proliferation effect wasmonitored by measuring the amount of DNA synthesized. In order toexclude the cytotoxicity of prodigiosin, it was used at a concentrationof not more than 100 nM. The effect of prodigiosin on the proliferationof lymphocyte is shown in Table 2, below. In Table 2, the proliferationpercentages mean the proliferated amounts of prodigiosin-treatedlymphocytes relative to that of an non-treated group. As shown, thesuppression percentage effected by prodigiosin in amounts of 30-100 nMreaches up to 96-98% for the T lymphocyte induced by concanavalin Awhile the proliferation of B lymphocyte induced by lipopolysaccharideand the proliferation of B/T lymphocytes induced by pokeweed mitogen aresuppressed to the extent of 13-19% and 45-83%, respectively.Consequently, the data demonstrate that the prodigiosin of the presentinvention has a potential immunosuppressive activity which is exertedselectively on T lymphocytes.

TABLE 2 Effect of Prodigiosin on the Proliferation of Immune Cells Conc.of Proliferation (%) Prodigiosin B/T Groups (nM) B cell T cell cellsNon- 100 100 100 treated Treated 3 101 77 100 10 105 46 86 30 87 4 55100 81 2 17

EXAMPLE IV

In vitro Experiment for the Effect of Prodigiosin on the Immune Response

The influence of prodigiosin on the functions of lymphocytes wasmeasured using three reaction systems. First, the ability of Blymphocyte to proliferate in response to lipopolysaccharide stimulus wasassessed. For this, on the third day after stimulation withlipopolysaccharide, the antibody production of the B lymphocyte wasmeasured. When B lymphocytes are stimulated with lipopolysaccharide,they can produce antibodies without the aid of T lymphocyte. Second, amixed lymphocyte reaction was induced in order to assess the effect onT-cell response. The reaction needs no aids from the B lymphocyte. Onthe third day after two types of heteroimmune cells, which are differentfrom each other in histocompatibility antigen, were mixed to stimulatethe activity of T lymphocytes, the T-cell response was assessed. Third,the T-cell dependent antibody producing reaction was utilized to assessthe effect of prodigiosin on the simultaneous immune response of both ofthe B and T lymphocytes. This reaction requires the functions of B and Tlymphocytes, simultaneously. On the fifth day after immunization of thelymphocytes with the red blood cells of sheep, their antibody productionability was assessed.

The effects of prodigiosin on the immune response of lymphocytes areshown in Table 3, below. As apparent from Table 3, the immune responsein which T lymphocytes are involved is significantly suppressed whereasthe B cell response is not at all throughout the concentration range. InTable 3, the values are relative to the immune response of thelymphocytes untreated with prodigiosin.

Taken together, the data of Examples III and IV demonstrate that theprodigiosin potentially suppresses the proliferation and immune responseof T lymphocytes, selectively.

TABLE 3 Effect of Prodigiosin on the Immune Response of Immune CellsConc. of Immune Response (%) Prodigiosin B/T Groups (nM) B cell T cellcells Non- 100 100 100 treated Treated 3 116 111 81 10 108 110 74 30 10067 64 100 97 30 34

EXAMPLE V

Selective Cytotoxicity of Prodiaiosin for B, CD4 T and CD8 T Lymphocytes

Whether the selective immunosuppression of prodigiosin for T cells isattributed to the selective cytotoxicity for T cells or not was assayedby measuring the proportion of the cells. On the third day aftertreatment of the immune cells with prodigiosin, the number of the cellswas counted. Because T lymphocytes consist of CD4 T cell (helper T cell)and CD8 T cell (cytotoxic T cell), the proportion of T and B lymphocyteswas calculated in this Example. The results are shown in Table 4, below.The data of Table 4 show that the prodigiosin has no selectivecytotoxicity. Thus, the selective immunosuppression for T lymphocytes isproved to be attributed to the suppression of immune response, but notof cytotoxicity. This result, together with the result of Example II,also demonstrates that the prodigiosin is not toxic within an effectiveexperimental concentration range.

TABLE 4 Cytotoxicity of Prodigiosin on Lymphocytes Conc. ofProliferation (%) Prodigiosin B CD4 T CD8 T Groups (nM) cell cell cellNon- 47 31 12 treated Treated 3 47 31 13 10 49 31 13 30 50 31 12 100 5229 10

EXAMPLE VI

In vivo Experiment for the Effect of Prodigiosin on T Lymphocyte

A graft versus host reaction was utilized for the in vivo assay ofprodigiosin's immunosuppression. The graft versus host reaction enablesan assessment of the immune response of T lymphocytes. On the sixth dayafter transplantation of the T lymphocytes of BDF1 mice different inhistocompatibility antigen, the lymphatic nodes were measured forweight, thereby assessing the immune response of T lymphocyte to thegrafted heteroantigens. The prodigiosin was peritoneally injected at adose of 30-100 mg per kg of body weight for five days whilecyclophosphamide, as a positive control, was peritoneally injected at adose of 100 mg/kg for five days. The body weights of the injected micewere measured to compare the toxicity of prodigiosin with that ofcyclophosphamide. The results are given in Table 5, providing testimonythat the prodigiosin potentially suppress the immune response of Tlymphocytes, like the positive control, cyclophosphamide. As for thebody weight, it was not changed in the mice injected with prodigiosin atan effective concentration. This demonstrates that the prodigiosinsuppresses the immune response of T lymphocyte without exerting toxicityin vivo. In contrast, a loss of body weight occurred in the miceinjected with cyclophosphamide at an effective concentration, showingthe toxicity of the chemical.

TABLE 5 Effect of Prodigiosin on T Lymphocyte Wt. (mg) of Body Conc.Lymphatic weight Groups (mg/kg) node (g) Prodigiosin non- 3.54 22treated Prodigiosin Treated 10 1.12 20 30 0.98 21 Positive Control 1000.06 18 (Cyclophosphamide)

EXAMPLE VII

Effect of Prodigiosin on T Lymphocytes in vivo (T-Cell Dependent ImmuneResponse)

A T cell-dependent immune response reaction was used to assess theinfluence of prodigiosin on T lymphocytes in vivo. Test animals wereimmunized with sheep red blood cells by peritoneal injection. 4 daysafter the immunization, the number of the antibody producing cells wascounted. Prodigiosin was peritoneally injected everyday. Based on thenumber of the antigen-producing cells in the non-treated animals, theinfluence of prodigiosin on T lymphocytes in vivo was assessed aspercentage. Also, the weight ratio of the spleen to the body wasmeasured to assay the toxicity of prodigiosin to the animals.Cyclophosphamide was used as a positive control.

The results are given in Table 6, below. As apparent from the data ofTable 6, the number of the antibody-producing cells was significantlyreduced by the treatment of prodigiosin, which is comparable to thepositive control, cyclophosphamide, in the immunosuppression. Takingaccount of the weight ratio of the spleen to the body, the prodigiosinshowed no toxicity at its effective concentrations whilecyclophosphamide was very toxic at its effective concentration.

TABLE 6 Effect of Prodigiosin on T cell -Dependent Immune ResponseImmune Conc. Response Wt. Ratio of Groups (mg/kg) (%) spleen/body (%)Prodigiosin 100 100 Treated Prodigiosin non- 10 32 95 treated 30 27 84Positive Control 100 7 26 (Cyclophosphamide)

Industrial Applicability

As apparent from the data of the Examples, the prodigiosin of thepresent invention has a potentially suppressive effect on the immuneresponse of T lymphocytes, in vivo and in vitro, both. What is better,the prodigiosin shows no toxicity at its effective concentration ranges.Therefore, the prodigiosin of the present invention can be used as animmunosuppressive or a standard substance in various fields, includingthe treatment of the diseases requiring immunosuppression and the basicresearch for the diseases, the transplantation of organs or tissues, andthe immune cells.

What is claimed is:
 1. A method of using prodigiosin to suppress theimmune response of T lymphocytes in a mammal comprising the step of:administering to said mammal an effective amount of said prodigiosin tosuppress the immune response of T lymphocytes having the chemicalformula (I) of

wherein said prodigiosin has a molecular weight of 323 and is producedfrom Serratia marcescens B-1231 (Korean Collection for Type Cultures,Korean Research Institute of Bioscience and Biotechnology, Access No.KCTC 0386BP).
 2. A method according to claim 1 wherein prodigiosin isadministered within an amount of about 30 mg to about 100 mg per kg ofbody weight.
 3. A method of suppressing the immune response of Tlymphocytes in a patient for the transplantation of organs, tissues, orimmune cells comprising the step of: administering to said patient aneffective amount of prodigiosin to suppress the immune response of Tlymphocytes having the chemical formula (I) of

wherein said prodigiosin has a molecular weight of 323 and is producedfrom Serratia marcescens B-1231 (Korean Collection for Type Cultures,Korean Research Institute of Bioscience and Biotechnology, Access No.KCTC 0386BP).
 4. A method according to claim 3 wherein prodigiosin isadministered within an amount of about 30 mg to about 100 mg per kg ofbody weight.